The distribution of rolling neutrophils in venular convergences.

The aim of this study was to characterize the distribution of adherent leukocytes in branched venular convergences in vivo. Intravital microscopy was used to obtain video images of leukocyte adhesion in multiple branched sites in mouse cremaster muscle, during the mild inflammatory response induced by surgical preparation. The average number of cells/vessel length was obtained over several minutes for seven venular convergences with varying geometrical configurations. Results from this study demonstrate a strong tendency of leukocytes to adhere at junctional points between converging vessels. Different vessel configurations were studied and results were shown to be insensitive to precise vessel geometry. Thus, in post-capillary venules, leukocytes are most likely to adhere at points between converging vessels, regardless of the precise geometrical properties or configuration of the vessels. Hydrodynamic mechanisms due to flow behavior through convergences likely play a significant role in determining locations of cellular adhesion. Future work should concentrate on quantifying the relative contributions of hydrodynamic and biochemical mechanisms to aid in understanding disease processes and development of treatments or therapeutics.

Coordinated redistribution of leukocyte LFA-1 and endothelial cell ICAM-1 accompany neutrophil transmigration.

The leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) and its endothelial ligand intercellular adhesion molecule (ICAM)-1 play an important role in transmigration as demonstrated by in vivo and in vitro models of inflammation. Despite the prominent role, little is known concerning the distribution and dynamic behavior of these adhesion molecules during leukocyte transmigration. Therefore, we examined the spatial and temporal distribution of LFA-1 on neutrophils actively transmigrating tumor necrosis factor-alpha-activated human umbilical vein endothelial monolayers under shear flow. Upon neutrophil arrest, LFA-1 was evenly distributed. However, once neutrophils initiated transmigration, LFA-1 rapidly redistributed to form a ringlike cluster at the neutrophil-endothelial junctional interface through which transmigration occurred. As transmigration was completed, LFA-1 redistributed to the neutrophil uropod. Endothelial ICAM-1 and JAM-A both colocalized with the ringlike LFA-1 cluster. Further analysis of PMA-stimulated neutrophils, which increase mobility of LFA-1, showed a rapid redistribution of LFA-1 and ICAM-1, but not endothelial JAM-A. Thus, endothelial JAM-A does not appear to contribute to adhesion or transmigration in this system. This is the first demonstration that neutrophil LFA-1 rapidly redistributes to form a ringlike structure that coclusters with endothelial ICAM-1 as the neutrophil transmigrates.

Elastase release by transmigrating neutrophils deactivates endothelial-bound SDF-1alpha and attenuates subsequent T lymphocyte transendothelial migration.

Leukocyte trafficking to sites of inflammation follows a defined temporal pattern, and evidence suggests that initial neutrophil transendothelial migration modifies endothelial cell phenotype. We tested the hypothesis that preconditioning of human umbilical vein endothelial cells (HUVEC) by neutrophils would also modify the subsequent transendothelial migration of T lymphocytes across cytokine-stimulated HUVEC in an in vitro flow assay. Using fluorescence microscopy, preconditioning of HUVEC by neutrophils was observed to significantly reduce the extent of subsequent stromal cell-derived factor-1alpha (SDF-1alpha [CXCL12])-mediated T lymphocyte transendothelial migration, without reducing accumulation. In contrast, recruitment of a second wave of neutrophils was unaltered. Conditioned medium harvested after transendothelial migration of neutrophils or supernatants from stimulated neutrophils mediated a similar blocking effect, which was negated using a specific neutrophil elastase inhibitor. Furthermore, T lymphocyte transendothelial migration was inhibited by treatment of HUVEC with purified neutrophil elastase, which selectively cleaved the amino terminus of HUVEC-bound SDF-1alpha, which is required for its chemotactic activity. The reduction in T lymphocyte transendothelial migration was not observed using a different chemokine, ELC (CCL19), and was not reversed by replenishment of SDF-1alpha, indicating endothelial retention of the inactivated chemokine. In summary, transmigrating neutrophils secrete localized elastase that is protected from plasma inhibitors, and thereby modulate trafficking of other leukocyte subsets by altering the endothelial-associated chemotactic activities.

Role of shear forces and adhesion molecule distribution on P-selectin-mediated leukocyte rolling in postcapillary venules.

Rolling on the venular endothelium is a critical step in the recruitment of leukocytes during the inflammatory response. P-selectin is a key mediator of leukocyte rolling, which is an early event in the inflammatory cascade; this rolling is likely to be directly regulated by both local fluid shear forces and P-selectin site densities in the microvasculature. However, neither the spatial pattern of P-selectin expression in postcapillary venules nor the effect of local expression patterns on rolling behavior in intact functional venules is known. We investigated the influence of local shear forces and the spatial distribution of endothelial P-selectin in intact blood perfused post capillary venules in anesthetized mice using intravital confocal microscopy, high temporal resolution particle tracking, and immunofluorescent labeling. We demonstrated a shear-dependent increase in average leukocyte rolling velocity that was attributable to a shear-dependent increase in the occurrence of transient leukocyte detachments from the endothelial surface: translational velocity during leukocyte contact with the vessel wall remained constant. P-selectin expression was not different in venules with characteristically different shear rates or diameters but varied significantly within individual venules. In postcapillary venules, regions of high P-selectin expression correlated with regions of slow leukocyte rolling. Thus the characteristically variable leukocyte rolling in vivo is a function of the spatial heterogeneity in P-selectin expression. The study shows how the local hydrodynamic forces and the nonuniform pattern of P-selectin expression affect the behavior of interacting leukocytes, providing direct evidence for the local variation of adhesion molecule expression as a mechanism for the regulation of leukocyte recruitment.

Regulation of leukocyte recruitment by local wall shear rate and leukocyte delivery.

OBJECTIVE: To determine the influence of wall shear rate and leukocyte delivery on leukocyte margination, rolling, and adhesion in post-capillary venules. METHODS: Leukocyte-endothelial cell interactions were characterized in cremaster muscle venules of anesthetized mice with video microscopy under control conditions and after 3 h exposure to TNF-alpha. Hemodynamic parameters were measured with fluorescent particle tracking using confocal microscopy. RESULTS: Leukocyte recruitment to the vessel wall and leukocyte rolling increased as a function of wall shear rate (P < 0.05) over the range observed (0-200 s(-1)) in intact post capillary venules. Leukocyte delivery affected recruitment and rolling flux only in activated vessels (P < 0.05). In addition, leukocyte firm adhesion was independent of both wall shear rate and leukocyte delivery, but showed an overall increase in TNF-alpha stimulated tissues (0.9 +/- 0.2 vs. 2.7 +/- 0.6 cells/50 microm). CONCLUSIONS: Leukocytes are delivered to venules in excess of the capacity of the local endothelium to support interactions. Elevated shear forces increase leukocyte recruitment to the vessel wall, which correlates to elevated rolling flux. In contrast, leukocyte firm adhesion is primarily affected by the activation state of the tissue and not by hemodynamic factors. Overall, the capacity of endothelial cells to support leukocyte interactions primarily regulates leukocyte recruitment and is not limited by leukocyte supply.

The effect of hematocrit and leukocyte adherence on flow direction in the microcirculation.

We sought to characterize how adherent leukocytes at the vessel wall, and the presence of erythrocytes, alter the streamlines (paths) of blood flow in the postcapillary venules. We directly visualized blood flow and leukocyte-endothelial cell interactions in postcapillary venules located in the cremaster muscle of anesthetized mice. Fluid streamlines were visualized by perfusing the cremaster muscle tissue with 0.5-micron fluorescent beads suspended in either buffer or whole blood, to examine the effect that erythrocytes have on the directionality of flow. Acute inflammation was induced in some animals by pretreatment of the vessels with tumor necrosis factor-alpha. To quantify the flow direction, the average deflection angle was defined as a scalar metric. Tracer bead trajectories were measurably altered by the presence of systemic levels of hematocrit, determined in each animal to be about 45%. Deviation from undirectional flow was also found to: (i) decrease with increasing vessel diameter, and (ii) increase with the number of adherent leukocytes. Fluid streamlines in the presence or absence of leukocyte adhesion or red cells agreed qualitatively with those obtained from theoretical calculations of blood flow using multiparticle adhesive dynamics. The microscale characteristics of venular flow are significantly altered during inflammation or changes in local hematocrit.

Hydrodynamic interactions between rolling leukocytes in vivo.

OBJECTIVE: The aim of this study was to characterize the hydrodynamic interactions between rolling and free-stream leukocytes in an in vivo model of selectin-mediated rolling, and to identify those physical mechanisms that influence the dynamics of transient adhesion with the walls of postcapillary venules. METHODS: Postcapillary venules of diameter 22-37 microm in the cheek pouch of anesthetized hamsters were visualized using intravital microscopy, with selectin-mediated rolling occurring in response to surgical preparation. RESULTS: Rolling velocity was found to be a strong function of the center-to-center separation with the nearest cell, and also was found to correlate strongly with the number of nearby cells. These effects are shown to be beyond that attributable to variations along the length of the vessel. Adherent leukocytes were observed to provide a nucleation site, precipitating further adhesion events of free-stream cells. CONCLUSIONS: The dynamics of the transient adhesion of leukocytes to the vessel wall in postcapillary venules is strongly dependent on the local concentration of adherent leukocytes, due to the complex hydrodynamics induced by their presence. The results are shown to agree well with theoretical considerations of the flow field induced by multiple nearby cells, suggesting a need for the future exploration of multicellular effects in the microcirculation.

Distributions of wall shear stress in venular convergences of mouse cremaster muscle.

OBJECTIVE: Wall shear stress regulates a variety of vascular functions and can be affected by variations in blood viscosity and wall shear rate independently. Therefore, the distribution of wall shear stress was characterized in converging flow regions of postcapillary venules to identify the relative contributions of shear rate and viscosity to wall shear rate and to determine whether venular branching directly affects the local shear environment. METHODS: Blood viscosity was evaluated as a function of vessel diameter and hematocrit, determined from fluorescent red blood cell (RBC) flux. Wall shear rate was measured directly from velocity profiles acquired using fluorescent RBC tracking or was estimated assuming Poiseuille flow conditions. Wall shear stress was calculated for measurements made across regions of converging flow. RESULTS: Measurements of wall shear stress showed no significant variation with location across the convergence (p < 0.9). Measures of viscosity showed little variation, for local measures of hematocrit (2.9 +/- 0.1 cp; coefficient of variation [CV]: 12%) or systemic hematocrit (3.4 +/- 0.1 centipoise [cp]; CV: 14%). Measures of wall shear rate showed greater variability for both direct measurements (61.2 +/- 8.7s; CV: 92%) and estimates assuming Poiseuille flow (122.7 +/- 15.7 s; CV: 83%). CONCLUSIONS: Variations in wall shear stress are independent of location through a venular convergence in contrast to arteriolar bifurcations. In addition, wall shear rate is significantly more variable than viscosity and, therefore, primarily accountable for wall shear stress variations in postcapillary venules.